Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-alpha2beta1 integrin antibody and accelerated by an alpha2beta1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-alpha5beta1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition.

Abstract-Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (nϭ9) were polygonal at confluence, 105Ϯ9 m in length, and had a doubling time of 39Ϯ2 hours. Spindle-shaped clones (nϭ3) were larger (267Ϯ18 m long, PϽ0.01) and grew slower (doubling time 65Ϯ4 hours, PϽ0.01). Both types of clones expressed smooth muscle (SM) ␣-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca 2ϩ transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM ␣-actin-positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior. (Circ Res. 2001;89:517-525.)

Background-The production of collagen is fundamental to atherosclerosis and critically dependent on posttranslational modification by prolyl 4-hydroxylase. Methods and Results-We report the cloning of a novel prolyl 4-hydroxylase catalytic (␣) subunit from human vascular smooth muscle cells. The peptide displayed conservation of critical residues for interacting with Fe 2ϩ and 2-oxoglutarate, essential cosubstrates for prolyl 4-hydroxylase activity. Furthermore, when the recombinant protein was expressed in cells, it associated with the ␤-subunit of prolyl 4-hydroxylase and could catalyze prolyl 4-hydroxylation of a collagen-like peptide. The tissue distribution was dissimilar from that of the 2 previously cloned ␣-subunits, suggesting a role beyond redundancy. Importantly, the novel gene was expressed in the fibrous cap of human carotid atherosclerotic lesions. Conclusions-The discovery of a novel prolyl 4-hydroxylase ␣-subunit, here termed the ␣(III)-subunit, suggests a new participant in collagen synthesis that, in view of the expression findings, may be relevant to atherosclerotic disease.